IL-1β Secretion In Response Research Articles

Gasdermin D and Gasdermin E Are Dispensable for Silica-Mediated IL-1β Secretion from Mouse Macrophages.

Silica crystals activate the NLRP3 inflammasome in macrophages, resulting in the caspase-1-dependent secretion of the proinflammatory cytokine IL-1β. Caspase-1-mediated cleavage of gasdermin D (GSDMD) triggers the formation of GSDMD pores, which drive pyroptotic cell death and facilitate the rapid release of IL-1β. However, the role of GSDMD in silica-induced lung injury is unclear. In this study, we show that although silica-induced lung injury is dependent on the inflammasome adaptor ASC and IL-1R1 signaling, GSDMD is dispensable for acute lung injury. Although the early rapid secretion of IL-1β in response to ATP and nigericin was GSDMD dependent, GSDMD was not required for IL-1β release at later time points. Similarly, secretion of IL-1β from macrophages in response to silica and alum proceeded in a GSDMD-independent manner. We further found that gasdermin E did not contribute to macrophage IL-1β secretion in the absence of GSDMD invitro and was also not necessary for silica-induced acute lung injury invivo. These findings demonstrate that GSDMD and gasdermin E are dispensable for IL-1β secretion in response to silica invitro and in silica-induced acute lung injury invivo.

TAT-beclin1 treatment accelerates the development of atherosclerotic lesions in ApoE-deficient mice.

The importance of autophagy in atherosclerosis has garnered significant attention regarding the potential applications of autophagy inducers. However, the impact of TAT-Beclin1, a peptide inducer of autophagy, on the development of atherosclerotic plaques remains unclear. Single-cell omics analysis indicates a notable reduction in GAPR1 levels within fibroblasts, stromal cells, and macrophages during atherosclerosis. Tat-beclin1 (T-B), an autophagy-inducing peptide derived from Beclin1, could selectively bind to GAPR1, relieving its inhibition on Beclin1 and thereby augmenting autophagosome formation. To investigate its impact on atherosclerosic plaque progression, we established the ApoE-/- mouse model of carotid atherosclerotic plaques. Surprisingly, intravenous administration of Tat-beclin1 dramatically accelerated the development of carotid artery plaques. Immunofluorescence analysis suggested that macrophage aggregation and autophagosome formation within atherosclerotic plaques were significantly increased upon T-B treatment. However, immunofluorescence and transmission electron microscopy (TEM) analysis revealed a reduction in autophagy flux through lysosomes. Invitro, the interaction between T-B and GAPR1 was confirmed in RAW264.7 cells, resulting in the increased accumulation of p62/SQSTM1 and LC3-II in the presence of ox-LDL. Additionally, T-B treatment elevated the protein levels of p62/SQSTM1, LC3-II, and cleaved caspase 1, along with the secretion of IL-1β in response to ox-LDL exposure. In summary, our study underscores that T-B treatment amplifies abnormal autophagy and inflammation, consequently exacerbating atherosclerotic plaque development in ApoE-/- mice.

P109 Organic Cation Transporter (OCTN)-1 variants shape innate immunity and predict individual response to vedolizumab In ulcerative colitis patients

Abstract Background Limited evidence is available about predictors of response to specific medications for ulcerative colitis (UC). OCTN1, an organic cation transporter, with its variants, could modulate the inflammatory response and potentially modify drug response to immune-modulatory therapy (anti-TNFα and vedolizumab), perhaps predicting individual response. Methods We first confirmed the ability of OCTN1 to differentially modulate IL1β secretion under different stimuli in a human leukemia monocytic cell line (THP-1) and in human peripheral blood mononuclear cells. We therefore prospectively enrolled a cohort of UC patients starting either anti-TNFα or vedolizumab, determined their OCTN1 genotype and evaluated their clinical features at baseline and clinical response to therapy or disease remission (according to clinical Mayo score) after six weeks (T1) and six months (T2). We finally enrolled a cohort of UC patients in clinical and endoscopic remission (according to full Mayo score) under immune-modulatory therapy (anti-TNFα or vedolizumab) for at least two years. Results OCTN1-deficient THP1 cells showed reduced IL-1β secretion in response to peptidoglycan (PGN) and Fusobacterium nucleatum (Fn), whilst primary monocytes bearing OCTN1 503F variant displayed an increased production of IL-1β in response to PGN and live bacteria. We therefore enrolled 90 patients, 50 starting anti-TNFα and 40 vedolizumab. Patients with 503F genotype showed higher rates of pancolitis as compared to 503L genotype. Among patients with 503L genotype, vedolizumab results highly associated with long-term response (OR 8.92, 95%CI 0.50-11.0; p=0.019), whilst conversely co-presence of 503F genotype and vedolizumab therapy disclosed an opposite role on the long-term response rate (OR 0.02, 95%CI 0.00-0.81; p=0.038).The population of UC patients in clinical and endoscopic remission was composed by 92 patients, 63 under anti-TNFα and 29 under vedolizumab. Of note, similar trends of disease extension were observed across genotypes: higher rates of pancolitis were observed in the 503F population as compared to 503L (62.5% vs 44%). Furthermore, among patients in remission with 503F genotype, the percentage of subjects receiving vedolizumab was sensibly lower than among patients with 503L genotype (87.5% anti-TNFα vs12.5% vedolizumab among 503F genotype; 56% anti-TNFα vs 44% vedolizumab among 503L genotype). Conclusion Mutated OCTN1 genotype (503F) favors an increased IL1β secretion from human stimulated leukocytes, thus suggesting a worse disease course with higher rate of pancolitis and lower response to vedolizumab as compared to 503L.

Characterization of Inflammasomes and Their Regulation in the Red Fox.

Inflammasomes recognize endogenous and exogenous danger signals, and subsequently induce the secretion of IL-1β. Studying inflammasomes in the red fox (Vulpes vulpes) is crucial for wildlife veterinary medicine, as it can help control inflammatory diseases in foxes. We investigated the activation and intracellular mechanisms of three inflammasomes (NLRP3, AIM2, and NLRC4) in fox peripheral blood mononuclear cells (PBMCs), using established triggers and inhibitors derived from humans and mice. Fox PBMCs exhibited normal activation and induction of IL-1β secretion in response to representative inflammasome triggers (ATP and nigericin for NLRP3, dsDNA for AIM2, flagellin for NLRC4). Additionally, PBMCs showed normal IL-1β secretion when inoculated with inflammasome-activating bacteria. In inhibitors of the inflammasome signaling pathway, fox inflammasome activation was compared with mouse inflammasomes. MCC950, a selective NLRP3 inhibitor, suppressed the secretion of dsDNA- and flagellin-mediated IL-1β in foxes, unlike mice. These findings suggest that NLRP3 may have a common role in dsDNA- and flagellin-mediated inflammasome activation in the red fox. It implies that this fox inflammasome biology can be applied to the treatment of inflammasome-mediated diseases in the red fox.

Mechanisms of systemic low-grade inflammation in HIV patients on long-term suppressive antiretroviral therapy: the inflammasome hypothesis.

We aimed to determine the contribution of inflammasome activation in chronic low-grade systemic inflammation observed in patients with HIV (PWH) on long-term suppressive antiretroviral therapy (ART) and to explore mechanisms of such activation. Forty-two PWH on long-term suppressive ART (HIV-RNA < 40 copies/ml) were compared with 10 HIV-negative healthy controls (HC). Inflammasome activation was measured by dosing mature interleukin (IL)-1β and IL-18 cytokines in patient serum. We explored inflammasome pathways through ex vivo stimulation of PWH primary monocytes with inflammasome activators; expression of inflammasome components by transcriptomic analysis; and metabolomics analysis of patient sera. Median (Q1; Q3) age, ART and viral suppression duration in PWH were 54 (48; 60), 15 (9; 20) and 7.5 (5; 12) years, respectively. Higher serum IL-18 was measured in PWH than in HC (61 (42; 77) vs. 36 (27-48 pg/ml), P = 0.009); IL-1β was detected in 10/42 PWH (0.5 (0.34; 0.80) pg/ml) but not in HC. Monocytes from PWH did not produce more inflammatory cytokines in vitro, but secretion of IL-1β in response to NLRP3 inflammasome stimulation was higher than in HC. This was not explained at the transcriptional level. We found an oxidative stress molecular profile in PWH sera. HIV infection with long-term effective ART is associated with a serum inflammatory signature, including markers of inflammasome activation, and an increased activation of monocytes upon inflammasome stimulation. Other cells should be investigated as sources of inflammatory cytokines in PWH. Oxidative stress might contribute to this chronic low-grade inflammation.

Ethanol extract of Chrysanthemumzawadskii inhibits the NLRP3 inflammasome by suppressing ASC oligomerization in macrophages.

Chrysanthemum zawadskii (C. zawadskii) is used in traditional East Asian medicine for the treatment of various diseases, including inflammatory disease. However, it has remained unclear whether extracts of C. zawadskii inhibit inflammasome activation in macrophages. The present study assessed the inhibitory effect of an ethanol extract of C. zawadskii (CZE) on the activation of the inflammasome in macrophages and the underlying mechanism. Bone marrow-derived macrophages were obtained from wild-type C57BL/6 mice. The release of IL-1β and lactate dehydrogenase in response to nucleotide-binding oligomerization domain-like receptor (NLR) family pyrin domain containing 3 (NLRP3) inflammasome activators, such as ATP, nigericin and monosodium urate (MSU) crystals, was significantly decreased by CZE in lipopolysaccharide (LPS)-primed BMDMs. Western blotting revealed that CZE inhibited ATP-induced caspase-1 cleavage and IL-1β maturation. To investigate whether CZE inhibits the priming step of the NLRP3 inflammasome, we confirmed the role of CZE at the gene level using RT-qPCR. CZE also downregulated the gene expression of NLRP3 and pro-IL-1β as well as NF-κB activation in BMDMs in response to LPS. Apoptosis-associated speck-like protein containing a caspase-recruitment domain (CARD) oligomerization and speck formation by NLRP3 inflammasome activators were suppressed by CZE. By contrast, CZE did not affect NLR family CARD domain-containing protein 4 or absent in melanoma 2 inflammasome activation in response to Salmonella typhimurium and poly(dA:dT) in LPS-primed BMDMs, respectively. The results revealed that three key components of CZE, namely linarin, 3,5-dicaffeoylquinic acid and chlorogenic acid, decreased IL-1β secretion in response to ATP, nigericin and MSU. These findings suggest that CZE effectively inhibited activation of the NLRP3 inflammasome.

IBD-associated G protein-coupled receptor 65 variant compromises signalling and impairs key functions involved in inflammation

Background and aimsInflammatory bowel diseases (IBD) result in chronic inflammation of the gastrointestinal tract. Genetic studies have shown that the GPR65 gene, as well as its missense coding variant, GPR65*Ile231Leu, is associated with IBD. We aimed to define the signalling and biological pathways downstream of GPR65 activation and evaluate the impact of GPR65*231Leu on these. MethodsWe used HEK 293 cells stably expressing GPR65 and deficient for either Gαs, Gαq/11 or Gα12/13, to define GPR65 signalling pathways, IBD patient biopsies and a panel of human tissues, primary immune cells and cell lines to determine biologic context, and genetic modulation of human THP-1-derived macrophages to examine the impact of GPR65 in bacterial phagocytosis and NLRP3 inflammasome activation. ResultsWe confirmed that GPR65 signals via the Gαs pathway, leading to cAMP accumulation. GPR65 can also signal via the Gα12/13 pathway leading to formation of stress fibers, actin remodeling and RhoA activation; all impaired by the IBD-associated GPR65*231Leu allele. Gene expression profiling revealed greater expression of GPR65 in biopsies from inflamed compared to non-inflamed tissues from IBD patients or control individuals, potentially explained by infiltration of inflammatory immune cells. Decreased GPR65 expression in THP-1-derived macrophages leads to impaired bacterial phagocytosis, increased NLRP3 inflammasome activation and IL-1β secretion in response to an inflammatory stimulus. ConclusionsWe demonstrate that GPR65 exerts its effects through Gαs- and Gα12/13-mediated pathways, that the IBD-associated GPR65*231Leu allele has compromised interactions with Gα12/13 and that KD of GPR65 leads to impaired bacterial phagocytosis and increased inflammatory signalling via the NLRP3 inflammasome. This work identifies a target for development of small molecule therapies.

Acetoacetate is a trigger of NLRP3 inflammasome activation in bovine peripheral blood mononuclear cells

Repeat breeding, which is non-pregnancy following three or more breeding attempts, is a serious reproductive disorder in cattle. In the present study, metabolomic profiling was used to identify metabolites in the blood plasma of repeat breeder cows (RBCs) and non-RBCs. Metabolomic analysis showed that acetoacetate (AcAc), a ketone body, was detected in RBCs, but not in non-RBCs. In contrast, β-hydroxybutyrate (BHB) was at similar levels in both RBCs and non-RBCs. We hypothesized that an imbalance of AcAc and BHB induces abnormal inflammatory conditions, especially the NLRP3 inflammasome, which regulates sterile inflammation to control interleukin (IL)-1β secretion, and may be associated with repeat breeding in cattle. To investigate this hypothesis, blood samples were collected from both non-RBCs and RBCs on day 7 of the estrous cycle. The mRNA expression of IL1B in peripheral blood mononuclear cells (PBMCs) was observed to be higher in RBCs than in non-RBCs. To test the effects of AcAc and BHB on inflammatory responses, blood samples were collected from healthy cows and PBMCs were isolated. PBMCs were treated with AcAc and BHB to investigate the activation of the NLRP3 inflammasome (complex of NLRP3, ASC, and caspase-1) and IL-1β secretion. AcAc treatment resulted in higher protein and/or mRNA expression of NLRP3 and IL-1β in PBMCs. Moreover, AcAc increased the co-localization of NLRP3 and ASC and stimulated caspase-1 activation, indicating the formation of the platform of the NLRP3 inflammasome. Addition of specific NLRP3 inhibitor, MCC950, suppressed AcAc stimulation-induced IL-1β secretion. Contrary to the effects of AcAc, BHB treatment suppressed the activation of NLRP3 inflammasome and IL-1β secretion in response to AcAc and typical NLRP3 inflammasome triggers. These findings demonstrate that AcAc can potentially trigger NLRP3 inflammasome activation, resulting in IL-1β secretion, and that these inflammatory responses are suppressed by BHB in bovine PBMCs. In addition, the imbalance between AcAc and BHB with higher levels of IL-1β may be associated with repeat breeding in cattle.

Korean Red Ginseng attenuates ultraviolet-mediated inflammasome activation in keratinocytes

BackgroundKeratinocytes form a physical barrier and act as an innate immune cell in skin. Keratinocytes secrete pro-inflammatory cytokines, such as interleukin (IL)-1β, resulting from inflammasome activation when exposed to ultraviolet (UV) irradiation. Korean Red Ginseng extracts (RGE) have been well-studied as modulators of inflammasome activation in immune cells, such as macrophages. In the study, we elucidated the role of RGE on the UV-mediated inflammasome activation in keratinocytes compared with that in macrophages. MethodsHuman skin keratinocyte cells (HaCaT), human epidermal keratinocytes (HEK), human monocyte-like cells (THP-1), and mouse macrophages were treated with RGE or a saponin fraction (SF) or non-saponin fraction (NS) of RGE before and after UV irradiation. The secretion levels of IL-1β, as an indicator of inflammasome activation, were analyzed. ResultsThe treatment of RGE or SF in macrophages after UV irradiation inhibited IL-1β secretion, but similar treatment in HaCaT cells did not. However, the treatment of RGE or SF in HaCaT cells in the presence of poly I:C, a toll-like receptor (TLR) 3 ligand, before UV exposure elicited the inhibition of the IL-1β secretion. The inhibition was caused by the disruption by RGE or SF of the TLR mediating up-regulation of the pro-IL-1β and NLRP3 genes during the priming step. ConclusionRGE and its saponins inhibit IL-1β secretion in response to UV exposure in both keratinocytes and macrophages. In particular, RGE treatment interrupted only the priming step in keratinocytes, although it did attenuate both the priming and activation steps in macrophages.

IκBζ regulates NLRP3 inflammasome activation

Abstract Inflammasome activation induces the maturation and secretion of interleukin (IL)-1β and -18, and is dependent on NF-κB signaling to induce the transcription of the inflammasome components, called the priming step. This study elucidated the role of IκBζ, an atypical IκBs (inhibitor of κB) and a coactivator of NF-κB target genes, on the activation of inflammasome. Bone marrow-derived macrophages (BMDMs) that originated from IκBζ-encoding Nfkbiz gene depletion mice presented a defect in NLRP3 inflammasome activation. In addition, the Nfkbiz+/− and Nfkbiz−/− mice significantly attenuated serum IL-1β secretion in response to a monosodium urate injection, a NLRP3 trigger, when compared with Nfkbiz−+/+ mice. The lack of IκBζ in BMDMs produced a disability in the expression of Nlrp3 and pro-Il1β mRNAs during the priming step. In addition, ectopic IκBζ expression enhanced the Nlrp3 promoter activity. Overall, IκBζ controlled the activation of NLRP3 inflammasome by upregulating the Nlrp3 gene during the priming step.

IκBζ controls NLRP3 inflammasome activation via upregulation of the Nlrp3 gene

Inflammasome activation induces the maturation and secretion of interleukin (IL)-1β and -18, and is dependent on NF-κB signaling to induce the transcription of the inflammasome components, called the priming step. This study elucidated the role of IκBζ, an atypical IκBs (inhibitor of κB) and a coactivator of NF-κB target genes, on the activation of inflammasome. Bone marrow-derived macrophages (BMDMs) that originated from IκBζ-encoding Nfkbiz gene depletion mice presented a defect in NLRP3 inflammasome activation. In addition, the Nfkbiz+/− and Nfkbiz−/− mice significantly attenuated serum IL-1β secretion in response to a monosodium urate injection, a NLRP3 trigger, when compared with Nfkbiz−+/+ mice. The lack of IκBζ in BMDMs produced a disability in the expression of Nlrp3 and pro-Il1β mRNAs during the priming step. In addition, ectopic IκBζ expression enhanced the Nlrp3 promoter activity, and Nlrp3 and pro-Il1β transcription. Overall, IκBζ controlled the activation of NLRP3 inflammasome by upregulating the Nlrp3 gene during the priming step.

NLRP3 inflammasome plays an important role in caspase-1 activation and IL-1β secretion in macrophages infected with Pasteurella multocida

Pasteurella multocida is a Gram-negative bacterium that is responsible for a variety of diseases in birds and mammals, including humans. We have previously reported that the P. multocida serotype A strain PmCQ2 causes severe lung pneumonia in bovines. Transcriptomic analysis showed that many genes related to the immune response were significantly upregulated in the lungs of mice infected with P. multocida compared with uninfected mice. However, the mechanism by which P. multocida induces host inflammatory cytokine secretion is poorly understood. In this study, the mechanism of caspase-1 activation and subsequent IL-1β secretion in macrophages infected with P. multocida was elucidated. The nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome was shown to be involved in inducing this cellular response. Compared with wild-type macrophages, Nlrp3−/− macrophages exhibited a clear decrease in caspase-1 activation and IL-1β secretion in response to P. multocida infection. Furthermore, spleen tyrosine kinase (Syk) was indicated to be involved in IL-1β secretion, possibly by regulating the NLRP3 inflammasome. Our results provide new insight into the host proinflammatory immune response against P. multocida and the critical involvement of the NLRP3 inflammasome in this activity.

Secretion of IL-1β From Monocytes in Gout Is Redox Independent.

The pro-inflammatory cytokine interleukin-1β (IL-1β) plays important roles in immunity but is also implicated in autoimmune disease. The most well-established mechanism of IL-1β secretion is via activation of the NOD-like receptor family pyrin domain containing-3 (NLRP3) inflammasome which requires an initial priming signal followed by an activating signal. However, the precise mechanism by which the inflammasome is activated remains unclear. The role of reactive oxygen species (ROS) in this process is contradictory, with some studies suggesting that ROS are crucial while others describe opposite effects. In this study, we evaluated the effects of oxidative stress on IL-1β secretion. Gout is a disease driven solely by IL-1β secretion in response to monosodium urate (MSU) crystals which form during periods of hyperuricemia and thus presents an opportunity to study factors contributing to IL-1β secretion. Sera and monocytes were isolated from patients with gout to determine whether differences in antioxidant status could explain the susceptibility of these individuals to gout attacks. In addition, sera and monocytes were collected from patients with chronic kidney disease (CKD) for comparison as this condition is associated with high levels of oxidative stress and disturbances in serum uric acid levels. There were differences in some aspects of antioxidant defenses in gout patients and these were mainly due to higher serum uric acid. Monocytes from gout patients were more responsive to priming, but not activation, of the NLRP3 inflammasome. However, expression of the components of the NLRP3 inflammasome were unaffected by priming or activation of the inflammasome, nor were these expression levels differentially regulated in gout patients. Inhibition of ROS by N-Acetyl Cysteine inhibited TLR2-induced priming of the NLRP3 inflammasome, but had no effect on MSU-induced activation. Together these findings demonstrate that oxidative stress only affects priming of the NLRP3 inflammasome but does not influence activation.

Characterisation of peripheral blood mononuclear cell populations in periparturient dairy cows that develop metritis

Bacterial contamination of the uterus following calving is ubiquitous in the dairy cow, 40% of cows develop postpartum uterine infection, including metritis. While predisposing factors like twinning and dystocia are associated with metritis, it is unclear why some cows remain healthy following calving and others develop uterine infection, negatively impacting animal health, milk production and economic return. Here, we profiled peripheral blood mononuclear cells of cows before calving and during postpartum metritis. We hypothesized that peripheral blood mononuclear cell function and proportions would be altered during the prepartum period in cows that develop postpartum metritis. Using flow cytometry we observed reduced proportions of peripheral CD3+/CD4+, CD4+/CD62L+, FOXP3+ and CD21+ populations from −10 to 40 days relative to calving associated with metritis, while the proportion of peripheral CD3+/CD4+ lymphocytes were specifically reduced in the prepartum period before the onset of metritis. Peripheral blood mononuclear cells from cows with metritis had a perturbed capacity to secrete IL-1β or IFNγ in response to in vitro stimulus; cells collected during the prepartum period from cows that would go on to develop metritis failed to increase IL-1β secretion in response to stimulation, while IFNγ secretion was altered at calving and postpartum in cows with metritis compared to healthy herd mates. No effect of metritis was observed in the capacity of cows to mount a humoral immune response to antigen administered on the day of calving. The studies discussed here suggest that while minor changes to the prepartum immune system are observed in cows that develop metritis, changes observed in the postpartum period are more prevalent and likely a consequences of disease and not causative. Future studies to modulate the prepartum immune system may help to limit postpartum metritis.

Characterization of Innate and Adaptive Immune Responses in PYNOD-Deficient Mice.

PYNOD (also called NLRP10) is a member of the nucleotide-binding domain and leucine-rich repeat containing family. Many members of this family play important roles in the activation and/or regulation of immune and inflammatory responses. We previously showed that PYNOD inhibits the IL-1β secretion in response to microbial infection in PYNOD-transgenic mice. In this study, we generated PYNOD-knockout (KO) mice and further investigated PYNOD's role in the innate and adaptive immune responses. Similar to wild-type macrophages, PYNOD-KO macrophages produced IL-1β and induced pyroptosis, a caspase-1-dependent programmed cell death, in response to various inflammasome activators and microbial infection. In addition, the PYNOD deficiency did not significantly affect the proliferation or cytokine production of T cells, the delayed-type hypersensitivity responses, the anti-tumor immunity, the Ag-specific Ab production, the cytotoxicity of NK cells, or the maturation, Ag-presenting capacity, or elicited migration of dendritic cells. Furthermore, the steady-state skin self-antigen transport to regional lymph nodes was not impaired in PYNOD-KO mice, suggesting that PYNOD is dispensable for steady-state dendritic cell migration. These results suggested that PYNOD is dispensable for the regulation of innate and adaptive immune responses in mice, unless PYNOD's expression is highly induced under certain conditions.

Pro-inflammatory adjuvant properties of pigment-grade titanium dioxide particles are augmented by a genotype that potentiates interleukin 1\u03b2 processing

BackgroundPigment-grade titanium dioxide (TiO2) particles are an additive to some foods (E171 on ingredients lists), toothpastes, and pharma−/nutraceuticals and are absorbed, to some extent, in the human intestinal tract. TiO2 can act as a modest adjuvant in the secretion of the pro-inflammatory cytokine interleukin 1β (IL-1β) when triggered by common intestinal bacterial fragments, such as lipopolysaccharide (LPS) and/or peptidoglycan.Given the variance in human genotypes, which includes variance in genes related to IL-1β secretion, we investigated whether TiO2 particles might, in fact, be more potent pro-inflammatory adjuvants in cells that are genetically susceptible to IL-1β-related inflammation.MethodsWe studied bone marrow-derived macrophages from mice with a mutation in the nucleotide-binding oligomerisation domain-containing 2 gene (Nod2m/m), which exhibit heightened secretion of IL-1β in response to the peptidoglycan fragment muramyl dipeptide (MDP). To ensure relevance to human exposure, TiO2 was food-grade anatase (119 ± 45 nm mean diameter ± standard deviation). We used a short ‘pulse and chase’ format: pulsing with LPS and chasing with TiO2 +/− MDP or peptidoglycan.ResultsIL-1β secretion was not stimulated in LPS-pulsed bone marrow-derived macrophages, or by chasing with MDP, and only very modestly so by chasing with peptidoglycan. In all cases, however, IL-1β secretion was augmented by chasing with TiO2 in a dose-dependent fashion (5–100 μg/mL). When co-administered with MDP or peptidoglycan, IL-1β secretion was further enhanced for the Nod2m/m genotype. Tumour necrosis factor α was triggered by LPS priming, and more so for the Nod2m/m genotype. This was enhanced by chasing with TiO2, MDP, or peptidoglycan, but there was no additive effect between the bacterial fragments and TiO2.ConclusionHere, the doses of TiO2 that augmented bacterial fragment-induced IL-1β secretion were relatively high. In vivo, however, selected intestinal cells appear to be loaded with TiO2, so such high concentrations may be ‘exposure-relevant’ for localised regions of the intestine where both TiO2 and bacterial fragment uptake occurs. Moreover, this effect is enhanced in cells from Nod2m/m mice indicating that genotype can dictate inflammatory signalling in response to (nano)particle exposure. In vivo studies are now merited.

Involvement of caspase-4 in IL-1 beta production and pyroptosis in human macrophages during dengue virus infection

Caspase-4 physically interacts with caspase-1 and is believed to be a proinflammatory caspase that can induce the inflammatory form of programmed cell death (pyroptosis) and the release of mature interleukin (IL)-1β. However, the function of caspase-4 in dengue virus infection is not yet fully understood. We examined the function of caspase-4 in IL-1β production and pyroptosis during dengue virus serotype-2 (DENV-2) infection in human macrophages. In this study, DENV-2 infection increased IL-1β protein level with activated caspase-4 activity. Using primary macrophages, we observed that caspase-4 induces activation of caspase-1 and secretion of IL-1β in response to DENV-2 infection, without the need for secondary signals to stimulate the assembly of the inflammasome. These findings indicate that the regulation of caspase-1 activity by capsase-4 could represent a unique mechanism. Our data suggest that caspase-4 is upstream of caspase-1 in the pathway that regulates pyroptosis and IL-1β synthesis in macrophages during DENV-2 infection.

LRRK2 promotes the activation of NLRC4 inflammasome during Salmonella Typhimurium infection.

Although genetic polymorphisms in the LRRK2 gene are associated with a variety of diseases, the physiological function of LRRK2 remains poorly understood. In this study, we report a crucial role for LRRK2 in the activation of the NLRC4 inflammasome during host defense against Salmonella enteric serovar Typhimurium infection. LRRK2 deficiency reduced caspase-1 activation and IL-1β secretion in response to NLRC4 inflammasome activators in macrophages. Lrrk2-/- mice exhibited impaired clearance of pathogens after acute S. Typhimurium infection. Mechanistically, LRRK2 formed a complex with NLRC4 in the macrophages, and the formation of the LRRK2-NLRC4 complex led to the phosphorylation of NLRC4 at Ser533. Importantly, the kinase activity of LRRK2 is required for optimal NLRC4 inflammasome activation. Collectively, our study reveals an important role for LRRK2 in the host defense by promoting NLRC4 inflammasome activation.

Implication of REDD1 in the activation of inflammatory pathways

In response to endotoxemia, the organism triggers an inflammatory response, and the visceral adipose tissue represents a major source of proinflammatory cytokines. The regulation of inflammation response in the adipose tissue is thus of crucial importance. We demonstrated that Regulated in development and DNA damage response-1 (REDD1) is involved in inflammation. REDD1 expression was increased in response to lipopolysaccharide (LPS) in bone marrow derived macrophages (BMDM) and in epidydimal adipose tissue. Loss of REDD1 protected the development of inflammation, since the expression of proinflammatory cytokines (TNFα, IL-6, IL-1β) was decreased in adipose tissue of REDD1−/− mice injected with LPS compared to wild-type mice. This decrease was associated with an inhibition of the activation of p38MAPK, JNK, NF-κB and NLRP3 inflammasome leading to a reduction of IL-1β secretion in response to LPS and ATP in REDD1−/− BMDM. Although REDD1 is an inhibitor of mTORC1, loss of REDD1 decreased inflammation independently of mTORC1 activation but more likely through oxidative stress regulation. Absence of REDD1 decreases ROS associated with a dysregulation of Nox-1 and GPx3 expression. Absence of REDD1 in macrophages decreases the development of insulin resistance in adipocyte-macrophage coculture. Altogether, REDD1 appears to be a key player in the control of inflammation.

Oxidized Low-Density Lipoprotein Immune Complex Priming of the Nlrp3 Inflammasome Involves TLR and FcγR Cooperation and Is Dependent on CARD9.

Oxidized low-density lipoprotein (oxLDL) is known to activate inflammatory responses in a variety of cells, especially macrophages and dendritic cells. Interestingly, much of the oxLDL in circulation is complexed to Abs, and these resulting immune complexes (ICs) are a prominent feature of chronic inflammatory disease, such as atherosclerosis, type-2 diabetes, systemic lupus erythematosus, and rheumatoid arthritis. Levels of oxLDL ICs often correlate with disease severity, and studies demonstrated that oxLDL ICs elicit potent inflammatory responses in macrophages. In this article, we show that bone marrow-derived dendritic cells (BMDCs) incubated with oxLDL ICs for 24 h secrete significantly more IL-1β compared with BMDCs treated with free oxLDL, whereas there was no difference in levels of TNF-α or IL-6. Treatment of BMDCs with oxLDL ICs increased expression of inflammasome-related genes Il1a, Il1b, and Nlrp3, and pretreatment with a caspase 1 inhibitor decreased IL-1β secretion in response to oxLDL ICs. This inflammasome priming was due to oxLDL IC signaling via multiple receptors, because inhibition of CD36, TLR4, and FcγR significantly decreased IL-1β secretion in response to oxLDL ICs. Signaling through these receptors converged on the adaptor protein CARD9, a component of the CARD9-Bcl10-MALT1 signalosome complex involved in NF-κB translocation. Finally, oxLDL IC-mediated IL-1β production resulted in increased Th17 polarization and cytokine secretion. Collectively, these data demonstrate that oxLDL ICs induce inflammasome activation through a separate and more robust mechanism than oxLDL alone and that these ICs may be immunomodulatory in chronic disease and not just biomarkers of severity.